UC San Diego

Drug discovery in the last century has largely been dominated by therapeutics targeting proteins. RNA targeting has remained largely elusive and high-risk, but some recent therapeutics have seen great utility and success. One area where RNA targeting has strong potential is in tackling viral infections. Many viruses, especially RNA viruses have high rates of mutation that sometimes confer treatment resistance. A method to counter the high mutation rates of viruses is to develop treatments which target conserved regions of the viral genome that are highly structured. For my studies, two structured RNA targets were probed: the Zika virus (ZIKV) stem loop A (SLA) and the SARS-CoV-2 RNA pseudoknot. Replication of mosquito-borne pathogenic flaviviruses like Zika virus depends on a noncoding RNA motif in the 5’ untranslated region of the genome which recruits the viral NS5 replicase. The three-dimensional architecture of the replication promoter RNA is termed the SLA. Sequence alignment as well as conservation and covariation analyses as evidence support a canonical RNA three-way junction (3WJ) architecture for the SLA RNA. The SLA 3WJ also influences viral translation by interaction with the E protein coding region and the conformational flexibility of the SLA 3WJ may be exploited to discover selectively binding ligands that interfere with viral replication. As a proof of concept, antisense DNA ligands hybridized at a domain of the SLA RNA served as surrogate ligands eliciting an extensive conformational change in the 3WJ fold which is readily detected by measuring FRET response. The FRET binding assay provides a useful tool for the discovery of small molecule ligands that target the flavivirus replication promoter. Another FRET binding assay was utilized to discover ligands which selectively bind the SARS-CoV-2 RNA pseudoknot. The discovered ligands consisted of aminoglycosides such as tobramycin and were retested with a phenotypic in vitro translation assay to assess for physiological relevance. While the results were not physiologically relevant, the discovered ligands act as a foundation for future work for discovering pan-SARS-CoV-2 mutant treatments and highlight the potential in targeting structured RNA as a drug target.